Difference between revisions of "Team:Paris Bettencourt/Project/Indigo"

Making patterns on denim

Abstract

The main focus of our project this year was enzyme design and discovery. We chose to engineer enzymes because they are a practical technology for removing stains, already known to be safe and economic. However, in our search for microbes that produce stain-removing enzymes we discovered something else: microbes are beautiful. When nutrients are added to fabric samples, bacterial and fungal colonies bloom in every size, shape and color. These microbes grow in beautiful patterns, remove pigments from fabric and leave other pigments behind. When the microbes were gone, the color and texture of the fabric was changed in ways that looked cool to us. What we describe below is not necessarily practical and only somewhat scientific. We are playing around with microbes on ordinary denim blue jeans and trying to create beauty.

Motivation and Background

Blue Jeans: Structure and Function

Jeans are trousers made with denim, a cotton textile. Blue jeans are dyed with indigo, a plant-derived pigment that is now mostly produced synthetically. In a classic blue denim weave, the warp thread is dyed with indigo while the weft threat is left white. The resulting fabric is blue on one surface, white on the other, with a grainy texture where the fibers cross. The blue threads are stained only on the outside and stay white at the core. As a result, the fabric lightens with wear, producing a characteristic fading pattern (Figure 1).

Denim Washes

The popular appeal of faded denim has lead to the development of many industrial processes that simulate or accelerate the fading process. New denim may be chemically treated, sandblasted, rubbed with sandpaper, or tumbled with abrasive stones prior to sale. These "wash" processes soften the fabric, fade the indigo, and produce a distinctive pattern and appearance. Especially with the trend toward high fashion and premium jeans, designers are motivated to discover new and innovative wash processes. The global jeans market was predicted to be 56 billion USD in 2014 (Agarwal, 2009).

Enzymatic Biowashing

In recent years, enzymes have become a popular tool for producing denim washes. Treatment with cellulases can degrade the external cellulose fibers under mild conditions (Montazer, 2008). As a result, it requires less energy, produces fewer chemical by-products, and causes less damage than chemical or abrasive washes.
Enzymatic treatment with laccase enzymes can be used to fade denim (Campos, 2001). Laccases (EC 1.10.3.2) are copper-containing oxidases that act on a variety of substrates. Acting on indigo, laccase abstracts one electron from each aromatic amine, leaving behind an unstable radical that quickly degrades.
Whole live cells with laccase activity have been used as bioremediation agents, in particular for indigo-containing wastewater from textile factories (Conceição, 2013). This activity was also the inspiration for the Bielefeld 2012 iGEM team. Thus, as we began this project, we knew that live cells and pure enzymes could act on the key components of blue jeans.

Results

We cultured microorganisms on denim covered with minimal M9, isolated the strains and identified them. Then, we tested their capacity at indigo’s degradation. At the end we have three microorganisms that degrade indigo: 2 Streptomyces and Pantoea.

Isolation of Microbes on Denim Media

In about one week we were able to isolate 16 strains of different colors and shapes on denim covered with media. Half strains are bacteria and the other half fungi. We identified them through 16S and 18S rRNA sequencing; however a few fungi are still unknown as we weren’t able to come with a suited protocol. We found mostly Aspergillus, Streptomyces and Enterobacter species. All our strains are shown in the table below. Confronted with what can be find in literature our results seem coherent: indeed Enterobacter are commonly found in bioreactors for waste and dye cleaning, and Aspergillus fumigatus and some Streptomyces were proved to degrade certain dyes, and specifically indigo in Streptomyces ‘case.

In a second time we wanted to make sure that these strains do eat indigo: as denim is made of cotton, the microbes we found could also use cellulose instead of indigo as carbon source, therefore not being of any interest for indigo fading on denim. Or not, cellulose are used in denim industry for washes? So we cultured all our strains on indigo plates, but only two were able to grow: Pantoea agglomerans P1 and Streptomyces S2. The problem with indigo plates is that we couldn’t make really blue plates, therefore we weren’t able to see a change of color on plates; the growth of strains was our only way of “assessing indigo degradation” using this methods.

Quantification of Indigo Degradation

To really quantify indigo degradation we tested our strains in liquid media so we could measure ODs. We proceeded in two rounds to select our best indigo eaters. We started with our 16 strains and after a few days the results allowed us to choose 8 strains as our candidates for the second round: F1, F2, F6, F7, S1, S3, S4 and P1. For all strains the OD was around 0,3 at Day 1, and at Day 4 we could really see which strains were degrading indigo faster than the other: we selected strains with OD under 0,07 and confirmed our choice with our own observations of indigo degradation in the tubes. Part of the results are shown in the graph below.

We carried out the second experiment with the 8 strains as said above, in the same conditions, and we did them in triplicates to collect sufficient data. After a week our second round gave us good results and we were able to choose 3 strains out of the 8 that we tested. For that we used the data we have and our own eyes as degradation was easy to notice: the liquid in the tubes containing these 3 strains had turned transparent, as opposed to the 5 other strains. By doing it in triplicates we could also make sure that our results were statistically significant, which they are. In the end, our three finalists are Streptomyces fumigatiscleroticus, Streptomyces coelicolor, Pantoea agglomerans. This is actually coherent with what we found with the indigo plates: Streptomyces and Pantoea agglomerans were the two strains that were able to grow on indigo plates. You can see the measures of the OD at Day 7 (last day) in the bar graph below.

Microbe Wash Denim

Laccases

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Methods

Preparation of Denim and Indigo Media

We used denim pieces cut out of a pair of jeans for our experiment. We put these pieces onto M9 minimal media agar supplemented with CaCL2 and MgSO4, but without a glucose carbon source. One piece was incubated in a bottle with the lid almost closed to avoid dehydration; the other pieces were put in onto square plates with minimal M9. After a few days different colonies were taken with an inoculation loop and inoculated on LB plates for growth.

Microbial Identification by rRNA Sequencing

To identify bacterial species, 16S sequences were amplified from isolated colonies by PCR. These samples were sequenced via Sanger sequencing (GATC Biotech), and identified through BLAST alignment (https://www.ncbi.nlm.nih.gov/BLAST/). The full protocol can be found here.

Fungi were identified through 18S rRNA sequencing. For each candidate species, one colony from an LB plate was inoculated on Sabouraud media and left to grow for 3 days. Genomic DNA was extracted using a DNeasy Blood and Tissue kit and a modified protocol, and was then used for 18S rRNA PCR (protocol can be found here).

Quantification of Indigo Consumption

Indigo plates : we used minimal M9 media and added indigo dissolved in DMSO, until blue color was visible in the plates. Candidate strains were then inoculated on M9-indigo plates and left to grow at 30°C.

Liquid M9 with indigo : 700µL of indigo in DMSO at 50mM was first added to 500mL Minimal M9 media. We put 5mL M9-indigo in 50 mL Falcon tubes, then put 20µL 50mM indigo in DMSO to have a deep blue color. Colonies from LB plates were used for inoculation. Tubes were left in a shaking incubator at 30°C for one week. Indigo Consumption was quantified using absorbance. Indigo strongest absorbance was determined at 680nm. Measurements were made every 24 hours for each strains for 6 days: 500µL media from previous 50mL falcon tubes were collected, vortexed, and 200µL were used in 96 well-plates. Absorbance was also measured at 450nm, for which indigo has a weak absorbance, to measure absorbance of cells and other things that can interfere with the absorbance at 680nm. Absorbance measurements were performed using a TECAN plate reader.

Stained cotton : small pieces of cotton were stained with around 100µL 50mM indigo dissolved in DMSO (sometimes more indigo was needed to completely stain the cotton). The cotton was washed at high temperatures, then sterilized with an ethanol wash. These pieces are then placed on M9 plates and liquid culture to assess indigo consumption on cotton.

Laccases

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Attributions

This project was done mostly by Mislav and Elisa. We would like to thank Jake for sacrifying his jeans for science, and XX for their collaboration.

References

• Agarwal, S. (2009). World Denim Market Production and Consuption Report 2012, Denimsandjeans.com.
• Montazer, M., & Maryan, A. S. (2008). Application of laccases with cellulases on denim for clean effluent and repeatable biowashing. Journal of Applied Polymer Science, 110(5), 3121–3129.
• Conceição, V., Freire, F. B., & Carvalho, K. Q. de. (2013). Treatment of textile effluent containing indigo blue dye by a UASB reactor coupled with pottery clay adsorption. Acta Scientiarum. Technology, 35(1), 1–6
• Campos, R., Kandelbauer, A., Robra, K. H., Cavaco-Paulo, A., & Gübitz, G. M. (2001). Indigo degradation with purified laccases from Trametes hirsuta and Sclerotium rolfsii. Journal of Biotechnology, 89(2-3), 131–139.
• Dubé, E., Shareck, F., Hurtubise, Y. et al. Appl Microbiol Biotechnol (2008) 79: 597. Homologous cloning, expression and characterization of a laccase from Streptomyces coelicolor and enzymatic decolourisation of an indigo dye.
• J Margot, C Bennati-Granier, J Maillard, P Blánquez, D.A Barry, C Holliger (2013). Bacterial versus fungal laccase : potential for micropollutant degradation. ABM Express ; 3 :63.
• Woodhead Publishing Series in Textile: Number 164; Denim, Manufacture, Finishing and Applications; edited by R. Paul